Antibodies and peptide antigens for producing antibodies having a selective binding specificity to bioactive intact parathyroid hormone (PTH) 1-84

ABSTRACT

Peptide antigens corresponding to amino acid residues 2-12, 1-12, 2-15 and 1-15 of parathyroid hormone (PTH), antibodies having an affinity to such peptide antigens and methods of producing the same. Such antigens, antibodies and methods producing the same according to the present invention are useful in determining bioactive intact PTH levels in serum, plasma, and/or cell culture media. Such antibodies further possess a high degree of species cross-reactivity, but substantially mitigated cross-reactivity to non-whole PTH peptide fragments and little to no recognition of the first amino acid residue of PTH.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 09/730,174, filed Dec. 5, 2000, entitled ANTIBODIESAND PEPTIDE ANTIGENS FOR PRODUCING ANTIBODIES HAVING A SELECTIVE BINDINGSPECIFICITY TO BIOACTIVE INTACT PARATHYROID HORMONE (PTH) 1-84, and nowin the issuance process, the teachings of which are expresslyincorporated herein by reference.

STATEMENT RE: FEDERALLY SPONSORED RESEARCH/DEVELOPMENT

Not Applicable

BACKGROUND OF THE INVENTION

Parathyroid hormone (PTH) and its importance in regulating theconcentration of calcium ions in the blood is well-known. In thisregard, such hormone is created by the parathyroid glands and, incombination with other factors, functions to regulate the blood calciumion levels such that the same is maintained in a steady concentration inboth cells and surrounding fluids. Essentially, PTH functions to releasestored calcium in the body when serum calcium levels decrease. On theother hand, such secretion is suppressed to the extent the serum calciumconcentration increases.

In its complete form PTH comprises a unique peptide comprised of 84amino acids. The specific sequence of PTH, as provided for a pluralityof species, namely, humans, rats, mice, bovids, dogs, pigs, cats andmonkeys are depicted in FIG. 1, and a variation thereof in FIG. 2, whichillustrates the relatively consistent structure such hormone maintainsbetween such identified species.

Given its significance in calcium metabolism for not only humans, but avariety of species, accurately measuring PTH has and continues to be ofsubstantial clinical significance. As is well-documented, serum PTHlevels serve as an important parameter for patients having diseases suchas hypercalcemia, primary hyperparathyroidism and osteoporosis, amongmany others. PTH likewise becomes of substantial clinical importance inpatients afflicted with chronic renal failure who, because of PTHabnormalities, can develop renal osteodystrophy.

Notwithstanding its important role in metabolism and clinicalsignificance, substantial difficulties have and continue to exist withregard to determinating circulating biologically active PTH levels.First of all, it is well-known that PTH is normally present at extremelylow levels, which are normally between 10 pg/ml to 65 pg/ml.Furthermore, it is known that the PTH peptide can be present in avariety of circulating PTH fragments, and in particular large non-(1-84)circulating PTH fragments which appear to co-migrate chromatographicallywith the (7-84) PTH molecule and are known to significantly interferewith conventional PTH assay measurements. Indeed, the large non-(1-84)PTH fragments may represent about one-half (½) of the PTH measured by amajority of current assays. Exemplary of the current shortcomings of theaccurate measurement of PTH are set forth in published PatentCooperation Treaty International Application No. PCT/US00/00855,International Publication No. W0/00/42437, entitled Methods forDifferentiating and Monitoring Parathyroid and Bone Status RelatedDiseases, and Lepage, Raymond et al., A Non-(1-84) CirculatingParathyroid Hormone (PTH) Fragment Interferes With Intact PTH CommercialAssay Measurement In Uremic Samples, Clinical Chemistry 44:4, 1998 pages805-809, the teachings of which are expressly incorporated herein byreference.

In an attempt to address such shortcomings, a new assay for detectingPTH levels was introduced by Scantibodies Laboratory, of Santee, Calif.,which incorporates a tracer antibody having a binding specificity forthe very end N-terminal fraction of human PTH, and more specifically,the first six amino acid residues thereof. As presently understood, suchassay appears to minimize cross-reactivity with large non-(1-84) PTHfragments. However, to derive such antibodies requires substantialeffort and expense in purifying the same. Moreover, such tracerantibodies have maximum recognition for only the first amino acidresidue of PTH, and substantially reduced specificity for any subsequentresidues thus obviating its use for some other species where the firstamino acid is different. Such drawbacks are discussed in the article byJohn, M. R. et al., entitled A Novel Immunoradiometric Assay DetectsFull-Length Human PTH but not Amino-Terminally Truncated Fragments:Implications for PTH Measurements in Renal Failure, The Journal ofClinical Endocrinology & Metabolism, Vol. 84, No. 11, 1999, p.4287-4290, the teachings of which are expressly incorporated herein byreference.

Thus, there has been and continues to be a long felt need in the art foran assay binding partner and method of generating the same that isspecific for bioactive intact PTH that can determine PTH levels withmitigated cross-reactivity to PTH peptide fragments. There is likewise aneed in the art for improved PTH binding partners that can measure PTHlevels in a more cost-effective manner and have a greater affinity forPTH that can be readily incorporated into immunoassay kits and the like.Still further there is a need for binding partners, namely, antibodieshaving a binding recognition that is specific to PTH and can be utilizedto detect PTH levels over a wide-variety of species. Finally, there is aneed in the art for an improved binding partner having a high bindingaffinity for PTH that may be readily derived using conventionalmechanisms that requires minimal purification, results in greaterbinding recognition for intact PTH, possesses minimal cross reactivityto large non-(1-84) PTH fragments, and can be derived utilizing methodsthat generate higher antibody yields than prior art binding partners.

BRIEF SUMMARY OF THE INVENTION

The present invention specifically addresses and alleviates theabove-identified deficiencies in art. In this regard, the presentinvention is directed to certain antigens, antibodies, and methods forproducing antibodies that are useful in determining bioactive intact PTHlevels in a sample fluid, such as serum, plasma or cell culture media.The antibodies and methods of the present invention have the particularadvantages of possessing greater affinity for PTH, and in particular,are designed to have a novel recognition for the first few amino acidresidues extending from the first N-terminal PTH residue but preferablynot beyond the fourth amino acid residue of PTH. In a most highlypreferred embodiment, the antibodies will have a specificity for thefirst three amino acid residues of N-terminal PTH. The antibodiesfurther have no cross-reactivity with the large non-(1-84) molecularforms of PTH. Moreover, the antigens, antibodies and methods ofproducing the same according to the present invention have substantialcross-reactivity with a wide variety of species, and may be utilized todetect PTH levels in not only humans, but also in rats, mice, bovids,dogs, pigs, cats and monkeys.

According to a preferred embodiment, the antigen comprises the formula:VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY

wherein X is selected from the group consisting of LEU [SEQ ID NO. 1]and PHE [SEQ ID NO. 2]. With respect to such embodiment, such antigenicpeptide represents amino acid residues 2-12 of PTH, with the sixth aminoacid residue thereof being selective between LEU and PHE, the formeroccurring in the PTH of humans, rats, mice and pigs, on one hand, andthe latter, being inherent in the PTH of bovines and dogs, on the otherhand. In a more highly preferred embodiment, the antigen comprises apeptide having the formula: Y-VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY

-   -   wherein X is an amino acid residue selected between LEU and PHE,        as discussed above, and Y is an amino acid residue consisting of        either SER or ALA [SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and        SEQ ID NO. 6, respectively], the former reflecting the amino        acid present in humans, dogs, and pigs, and the latter being        inherent in the PTH of rats, mice and bovines.

In further preferred embodiments, the antigen comprises the formula:VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY-LYS-HIS- LEU

wherein X is selected from the group consisting of LEU [SEQ ID NO. 7] orPHE [SEQ ID NO. 8]. Such antigenic peptide represents amino acidresidues 2-15 of PTH, with the sixth amino acid residue comprisingeither LEU or PHE, to thus reflect the corresponding amino acid residueoccurring in the appropriate species specified above. In anotherembodiment, the antigenic peptide represents amino acid residues 1-15 ofPTH and comprises the formula:Y-VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY-LYS- HIS-LEU

-   -   wherein X comprises amino acid residue LEU or PHE and Y is an        amino acid residue consisting of either SER or ALA [SEQ ID NO.        9, SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12,        respectively], the latter being selective to correspond to a        particular species identified above.

With respect to the antibodies and methods of producing the sameaccording to the present invention, such are directed to antibodieshaving an affinity and specificity for the aforementioned antigens.Preferably, the antibodies are specific to amino acid residues 2-12,1-12, 2-15 and 1-15, respectively, of PTH. In a most highly preferredembodiment, the antibodies are specific to only the first three aminoacid residues of PTH. Such antibodies are preferably produced via thesteps of administering a peptide antigen of the aforementioned variety,either alone or in combination with a carrier protein, to a host animal,which preferably comprises a goat, to produce antibody productionagainst the peptide antigen. In an alternative, preferred embodiment,antibody production is induced via the administration of larger peptidefragments of PTH. Preferably, such PTH fragment may comprise (1-34) PTH,which may further optionally include a carrier protein covalently linkedor fused thereto to increase antigenicity or intact (1-84) PTH. To theextent antibodies are sought to be derived to detect PTH in humans, theintact (1-84) PTH molecule preferably comprises intact rat PTH or, to alesser extent, human PTH. The antibody titer produced by theadministration of the antigen to the host animal is then monitored.Thereafter, antisera produced in the host animal is then isolated andthe antibodies thereof are selected by affinity chromatography to havespecificity for the desired antigenic region of PTH (i.e., amino acidresidues 2-12, 1-12, 2-15 and 1-15 of PTH, respectively). In anadditional step, the isolated antibodies are further purified to removethose antibodies having an affinity for amino acid residues extendingbeyond the fourth or fifth N-terminal PTH amino acid residue by usingbound antigens corresponding to either (4-34) PTH and/or (4-84) PTH or(5-34) PTH and/or (5-84) PTH that retain unwanted antibodies but allowthose antibodies specific for only (1-3) or (1-4) PTH to pass. Theantibodies may then be labeled or otherwise incorporated into any of avariety of conventional assays for use in the detection of PTH, whetherit would be for humans or any of a variety of species.

As will be recognized by those skilled in the art, the antigens,antibodies and methods of the present invention, by focusing on aminoacid residues 2-12, 1-12, 2-15 and 1-15 of PTH, respectively, focus onthat portion of the PTH molecule possessing N-terminal biologicalactivity, and thus maximize detection of the same. Moreover, withrespect to the more highly preferred embodiments, by providing antigens,antibodies and methods of producing the same that are inclusive of otheramino acid residues extending beyond the N-terminal biologically activesite (i.e., up to and including the twelfth (12th) and fifteenth (15th)amino acid residues of PTH), the specificity and affinity of suchantibodies are thus more highly refined and enable the same to detectPTH levels with greater specificity and affinity than prior artreceptors, as incorporated into assays and the like. In a most highlypreferred embodiment, the antibodies have a specificity for no more thanthe first three amino acid residues of PTH which, as discussed, isaccomplished via a removal or “scrubbing” step whereby antibodiesproduced via the use of the antigens of the present invention areselectively removed to the extent any such antibodies have an affinityfor (4-34) PTH or (4-84) PTH. As a consequence, the antibodies producedaccording to the present invention (as well as the methods and peptideantigens disclosed herein for achieving that end) have substantiallyeliminated cross-reactivity to large, non (1-84) PTH peptide fragments,do not possess maximum recognition for only the first amino acid residueof PTH, and further, may be readily derived in a cost-effective mannerinsofar as the antibody yields generated from methods of the presentinvention should be greater than prior art methods.

Alternatively, such removal or “scrubbing” step will be utilized toselectively remove antibodies having an affinity for (5-34) PTH or(5-84) PTH. By doing so, the antibodies ultimately isolated will have anaffinity for no more than the first four amino acid residues ofN-terminal PTH. It is therefore an object of the present invention toprovide: 1) select antigens for the production and isolation ofantibodies; 2) antibodies; and 3) methods of producing antibodies thathave a binding affinity and specificity for PTH that possess mitigatedcross-reactivity to large non (1-84) PTH peptide fragments.

Another object of the present invention is to provide: 1) selectantigens for the production of antibodies; 2) antibodies; and 3) methodsof producing antibodies that have a greater affinity and specificity forPTH than prior art binding partners and further, possess a higher degreeof cross reactivity between inter-species PTH such that the antigens,antibodies and methods producing the same according the presentinvention can be readily utilized for the detection of PTH in a varietyof species.

Another object of the present invention is to provide: 1) selectantigens for the production and isolation of antibodies; 2) antibodies;and 3) methods of producing antibodies which have a binding affinity andspecificity for more of the biologically active N-terminal portion ofPTH and, hence, are more effective and accurate in determining bioactiveintact PTH levels than prior art binding partners directed thereto.

Another object of the present invention is to provide: 1) selectantigens for the production and isolation of antibodies; 2) antibodies;and 3) methods of producing antibodies which do not possess a maximumbinding affinity for only the first N-terminal amino acid residue ofPTH.

Another object of the present invention is to provide: 1) selectantigens for the production and isolation of antibodies; 2) antibodies;and 3) methods of producing antibodies that are less expensive toproduce and generate higher antibody yields than prior art methods forproducing antibodies having a binding affinity and specificity for theN-terminal of PTH.

Still further objects of the present invention are to provide: 1) selectantigens for the production and isolation of antibodies; 2) antibodies;and 3) methods of producing antibodies which readily derive antibodieswhich may be readily incorporated into any of a variety ofcommercially-available assays and further, can be modified (e.g., toinclude a label, and the like) as may be desired for any of a variety ofimmunoassay applications.

BRIEF DESCRIPTION OF THE DRAWINGS

These, as well as other features of the present invention, will becomemore apparent upon reference to the drawings wherein:

FIG. 1 depicts the amino acid sequence of PTH for a variety of species,namely humans, rats, mice, bovines, dogs, pigs, felines and monkeys andfurther depicts amino acid sequences identified herein as SEQ ID NO. 1,SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6,SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11,and SEQ ID NO. 12.

FIG. 2 is an alternate illustration of FIG. 1 depicting a variation ofthe 1-84 amino acid sequence of PTH of the aforementioned species, yetdepicting the conserved N-terminus of PTH wherein the amino acidsequences of SEQ ID NOS. 1-12 remain relatively constant.

FIG. 3 is a diagrammatical view of the N-terminal portion of human PTH.

FIG. 4 is a flow chart depicting the steps for producing antibodiesaccording to a preferred embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The detailed description set forth below is intended as a description ofthe presently preferred embodiment of the invention, and is not intendedto represent the only form in which the present invention may beconstructed or utilized. The description sets forth the functions andsequences of steps for constructing and operating the invention. It isto be understood, however, that the same or equivalent functions andsequences may be accomplished by different embodiments and that they arealso intended to be encompassed within the scope of the invention.

The present invention encompasses antigens, antibodies and methods ofproducing antibodies that are directed to an antigenic region of PTHpositioned in the N-terminal region thereof, and more precisely, thefirst fifteen (15) amino acid residues extending from the N-terminaldepicted as 10 in FIGS. 1-3. In a most highly preferred embodiment, theantibodies are specific for only the first three (3) amino acid residuesof PTH. As is well-known, the N-terminal region of PTH is recognized asnecessary for PTH/PTHrp receptor binding, and is further recognized asbeing most desirable epitope for measuring bioactive intact PTH levels,as may be found in a biological fluid sample, such as serum, plasma orcell culture media. Indicative of the current state of the artassociated with PTH and the methods of detecting the same are discussedat length in published Patent Cooperation Treaty InternationalApplication No. PCT/US00/00855, International Publication No.W0/00/42437, entitled Methods for Differentiating and MonitoringParathyroid and Bone Status Related Diseases, and Lepage, Raymond etal., A Non-(1-84) Circulating Parathyroid Hormone (PTH) FragmentInterferes With Intact PTH Commercial Assay Measurement In UremicSamples, Clinical Chemistry 44:4, 1998 pages 805-809, the teachings ofwhich are expressly incorporated herein by reference.

According to a preferred embodiment, the antigenic peptide of thepresent invention comprises those amino acid residues corresponding toamino acid residues 2-12 of PTH, collectively identified as 12 in FIGS.1-3. Specifically, such antigenic peptide will have the formula:VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY

-   -   wherein X is an amino acid residue selected from either LEU [SEQ        NO. 1] or PHE [SEQ NO. 2]. As will be recognized by those        skilled in the art, the sixth (6th) amino acid residue of this        PTH peptide fragment does differ between the cited species        whereby such residue comprises LEU in humans, rats, mice and        pigs, on one hand, but PHE for bovids and dogs on the other. As        will be appreciated by those skilled in the art, notwithstanding        the single amino acid residue difference, such antigenic peptide        remains otherwise constant between the cited species which, as        discussed more fully below, can enable antibodies to be prepared        and ultimately utilized that are cross-reactive and, hence,        effective in detecting PTH levels in a variety of such species.

In a more highly preferred embodiment, the peptide antigen reflects thefirst twelve (12) amino acid residues of PTH, identified as 14, andcomprises the formula: Y-VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY

-   -   wherein X is an amino acid residue selected from either LEU or        PHE and Y is an amino acid residue selected from either SER or        ALA [SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6,        respectively]. With respect to the variation at the first amino        acid residue, it will be readily appreciated that such antigenic        peptide may be formed such that such amino acid comprises SER,        as found in humans, dogs and pigs, or ALA, as found in rats,        mice and bovids. In this respect, the variation provided for in        the antigenic peptide in the present invention, and in        particular the more highly preferred embodiments thereof,        provide leeway such that the antibodies ultimately derived from        such antigenic peptides may be formed to possess a higher        binding affinity as may be desired to detect PTH in a given        species.

In more highly refined embodiments of the present invention, theantigenic peptides comprise sequences that correspond to amino acidresidue 2-15 and 1-15, respectively, of PTH. With regard to the former,identified in FIGS. 1-3 as 16, such antigenic peptide will have theformula: VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY-LYS-HIS- LEU

wherein X is an amino acid residue selected from either LEU [SEQ ID NO.7] or PHE [SEQ ID NO. 8]. With respect to the latter embodimentcorresponding to amino acid residues 1-15 of PTH, identified as 10, suchantigenic peptide will have the formula:Y-VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY-LYS- HIS-LEU

-   -   wherein X is an amino acid residue selected from either LEU or        PHE and Y is an amino acid residue selected from either SER or        ALA [SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO.        12, respectively]. A compact disc providing a computer readable        form of SEQ ID NOS. 1-12 in compliance with the requirements of        37 CFR 1.821-1.825, which is identical to the written sequence        listing provided herein, has been submitted to comply with such        statutory requirements. Notwithstanding the foregoing formulas        for the aforementioned peptides, it will be recognized that the        same will extend to all functional derivatives thereof, which is        meant to include functionally comparable peptides derived from        the same region of PTH, as reflected in the sequences of FIGS.        1-3, and having a similar ability to induce specific anti-PTH        antibodies, and more particularly antibodies specific to the        N-terminal amino acid residues of PTH. In this regard, such        functional derivative may be similarly positioned peptides or        peptides derived from the sequences discussed above and        reflected in FIGS. 1-3 having substitutions, additions or        deletions of amino acids, provided the derivation does not alter        the ability of the peptide antigen to induce antibody reactive        to PTH.

It should further be recognized that the peptide antigens of the presentinvention include those peptides whose amino acid sequence may beshifted within a few amino acids upstream or downstream of the peptidesdiscussed above in FIGS. 1-3, as well as those peptides havingconservative amino acid changes such that substitutions, additions ordeletions of amino acids or changes do not significantly effect theability of the peptide antigen to induce antibodies with high affinityand specificity for the first twelve amino acid residues of PTH, or anysub-components thereof.

With regard to antibody production, there is illustrated in FIG. 4 themethod 20 of generating the same. Initially, there is provided a peptide22 of the aforementioned variety which corresponds to all or a portionof the first twelve (12) to fifteen (15) amino acid residues of PTH of agiven species. According to a more highly preferred embodiment, however,the antigenic peptide utilized to generate antibody production willinclude larger PTH fragments, including but not limited to the (1-34)PTH and the entire intact (1-84) PTH peptide. Along these lines, whilethe antibodies ultimately produced will have specificity to those aminoacid residues corresponding to the aforementioned antigenic peptides(i.e., amino acid sequencing 2-12, 1-12, 2-15 and/or 1-15 of PTH) withinthe method of the present invention, at least with respect to theinitial step of providing an antigen 22 and subsequently administeringthe same, via step 24 discussed below, it will be appreciated that theentire PTH peptide and any N-terminal fragment thereof can preferablyserve as a suitable antigenic peptide to induce the production ofantibodies which will ultimately possess the ideal binding affinity andspecificity for the biologically active portion of PTH at the N-terminalthereof. In a most highly preferred embodiment, the antigenic peptideprovided and administered will comprise the PTH fragment correspondingto amino acid residues 1-34 of PTH, which may optionally be coupled to acarrier protein. Such peptide fragment may alternatively preferably beadministered as whole length (1-84) PTH, which may take the form of anyof the species identified in FIGS. 1 and 2. Preferably, in the contextof developing antibodies specific for human PTH, the use of (1-34) ratPTH or whole length (1-84) rat PTH is preferred.

Such antigenic peptides may be produced by any of a variety of methodswell-known in the art, including synthesis by conventional methods, suchas solid-phase chemical synthesis or by recombinant technology. As willbe further appreciated, the synthetic peptides may optionally bechemically coupled to a carrier protein or, alternatively, recombinantpeptides may be generated as fusion proteins to increase antigenicity.As will be further appreciated by those skilled in the art, suchantigenic peptides may be screened based upon their ability to induceanti PTH antibody. In this respect, such screening techniques mayinclude, but are not limited to, immunoprecipitation or immunoassay.

Once derived, such peptide antigens may then be utilized to generate theantibodies of the present invention using conventional techniques. Inthis regard, the peptide antigen(s), preferably in combination with anadjuvant, is/are administered to a host animal 24, which preferablycomprises a goat. It should be recognized, however, that other species,such as rabbits, mice, sheep, chicken, etc., may additionally beutilized as host animals. In this respect, the administration of suchantigens may be accomplished by any of a variety of methods, includingbut not limited to subcutaneous or intramuscular injection. As will beappreciated, the dose of peptide antigen administered willcorrespondingly vary with the specific peptide utilized, as well as theanimal host. As will additionally be recognized, however, in order toobtain antibodies possessing the highest affinity and specificitypossible for a given species, separate antibodies should be generatedagainst the appropriate comparable peptide from each species.

Once administered, the results of antibody titers produced in the hostanimal are then monitored 26, which may be conducted by any of a varietyof techniques well-known in the art, using routine bleeds and the like,with the antisera being isolated (e.g., via centrifugation), in step 28,and thereafter screened for the presence of anti-peptide antibodieshaving a binding affinity therefor. It will further be recognized thatgiven the foregoing conventional immunological methods for deriving theantibodies of the present invention, such antibodies may be monoclonalor polyclonal in nature. Consistent with conventional practice it ispreferred that the antisera be derived from a plurality of host animals.

The resultant antisera derived from the host animal may be affinitypurified to derive the antibodies for the present invention. As iswell-known in the art, the antisera may be purified via conventionaltechniques, such as the introduction into a separation column with theaforementioned antigenic peptides corresponding to amino acid residuesequences 2-12, 1-12, 2-15 and/or 1-15 of PTH being bound to a solidphase (e.g., beads and the like). The antiserum may then be washed toremove antibodies not having specificity for the antigenic peptide orpeptides, with the remaining bound antibody, specific for the antigenicpeptide or peptides, ultimately being eluted therefrom. Such antibodymay then be stored per conventional practices well-known to thoseskilled in the art.

In an additional step 29, the antibodies derived through such isolationand purification process will undergo a further purification processwhereby any antibodies present in the antisera having an affinity for(4-34) or (5-34) PTH and/or (4-84) or (5-84) PTH are selectively removedor “scrubbed.” To that end, it is contemplated that once the antisera isisolated in step 28, the same may be purified to remove any antibodieshaving specificity for (4-34) or (5-34) PTH and/or (4-84) or (5-84) PTHfor any of the species specified herein via the use of a separationcolumn or other known conventional techniques whereby antigenic peptidescorresponding to either amino acid sequences (4-34) or (5-34) PTH or(4-84) (5-84) PTH are bound to a solid phase with the antisera sought tobe purified being subjected thereto. Any antibody having a specificityfor a specific (4-34) or (5-34) PTH and/or (4-84) or (5-84) PTH peptidewill remain bound and the remaining antibodies not having specificityfor the (4-34) or (5-34) PTH or (4-84) or (5-84) PTH peptide will beseparated therefrom. Advantageously, such process will thus isolate onlythose antibodies having an affinity for at most the first three (3) orfour (4) amino residues of PTH, and thus will have no binding affinitywhatsoever for any amino acid residues at or beyond the fifth amino acidresidue of the N-terminal of PTH. Such process thus ensures that anyantibodies ultimately isolated will, in fact, be specific to the N-mostterminal of (1-84) PTH and thus not otherwise bind to any type of PTHfragment that does not include at least the first three or fourN-terminus amino acid residues of PTH.

As such antibodies are isolated using those antigenic peptidescorresponding to amino acid residue sequences 2-12 and 2-15 of PTH,respectively, there will thus correspondingly be isolated antibodiesthat have not necessarily acquired a binding affinity, much less amaximum binding affinity, for the first amino acid residue of PTH, whichis known to be problematic of antibodies derived via prior art methods.Along these lines, the elimination or substantial suppression ofantibodies having an affinity for the first amino acid residue of PTHwill likewise be achieved by utilizing peptide sequences correspondingto 1-12 and 1-15, respectively, of PTH provided that such sequences areselected from those species having a dissimilar first amino acid residuefrom the species for which the antibodies ultimately derived will beutilized. For example, in order to derive antibodies suitable fordetecting PTH levels in humans that lack in specificity for the firstamino acid residue of human PTH, it will be recognized that anti-rat PTHsera derived from the host animal may be purified against peptidescorresponding to amino acid residue sequences 1-12 and 1-15 of humanPTH. As will be understood, because the first N-terminal amino acidresidue of rat PTH comprises ALA, as opposed to SER found in humans, anyantibodies ultimately isolated will necessarily possess a bindingaffinity for the amino acid residues extending beyond such first aminoacid residue sequence.

Once derivatized in step 30, such antibodies may be used inimmunological techniques to correlate the presence of bioactive intactPTH as may be found in a given sample (e.g., serum or plasma). In thisrespect, the antibodies of the present invention may be used alone or incombination to screen a given sample to determine the presence ofbioactive intact PTH but yet advantageously avoid cross-reactivity withthe large non (1-84) PTH fragments. By way of example, such antibodiesmay be incorporated into an immunological assay kit. Exemplary of suchapplications include the human bioactive intact PTH and rat bioactiveintact PTH ELISA kits, produced by Immutopics, Inc., of San Clemente,Calif. which provide an enzyme-linked immunosorbent assay (ELISA) forthe quantitative determination of bioactive intact PTH levels in serum,plasma or cell culture media. In this regard, given the foregoingapplicability to derive antibodies specific to PTH for a variety ofspecies, it will be recognized that such immunological assay kits, suchas those provided by Immutopics, Inc., may be specifically designed suchthat the affinity and specificity of such antibodies apply to a widevariety of species or alternatively, generated against the appropriatecomparable peptide of a given species such that the kits are morenarrowly tailored for a given species.

Additional modifications and improvements of the present invention mayalso be apparent to those of ordinary skill in the art. Thus, theparticular combination of parts described and illustrated herein isintended to represent only a certain embodiment of the presentinvention, and is not intended to serve as a limitation of alternativedevices within the spirit and scope of the invention.

1. A method for producing an antibody to the N-terminal portion of(1-84) PTH useful in the determination of intact PTH 1-84 levels in abiological sample, the method comprising the steps: a) administering afirst peptide antigen to a host animal to induce antibody productionagainst said first peptide antigen in said host animal, said firstpeptide antigen being selected from the group consisting of SEQ ID NO.3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, (1-34) PTH and (1-84) PTH;b) monitoring antibody titer produced by said administration of said atleast one antigen to said host animal; c) extracting antisera producedin said host animal by said administration of said at least one peptideantigen; d) isolating and selecting at least one antibody from saidantisera extracted in step c) by affinity chromatography utilizing asecond peptide antigen selected from the group consisting of SEQ ID NO.3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO.6; e) removing said atleast one antibody isolated in step d) to the extent said at least oneantibody is specific for a peptide antigen selected from the groupconsisting of (4-34) PTH, (5-34) PTH, (4-84) PTH and (5-84) PTH; and f)isolating said antibody in steps d) and e) to the extent said antibodyis not specific for said antigen selected from the group consisting of(4-34) PTH, (5-34) PTH, (4-84) PTH and (5-84) PTH.
 2. The method ofclaim 1 wherein in step a), said host animal is selected from the groupconsisting of a mouse and a rabbit.
 3. The method of claim 1 wherein instep a), said host animal comprises at least one goat.
 4. The method ofclaim 1 wherein in step a), said (1-34) PTH is derived from a speciesselected from the group consisting of a human, a rat, a mouse, a bovine,a dog, a pig, a cat, and a monkey.
 5. The method of claim 1 wherein instep a), said first peptide antigen has a carrier protein coupledtherewith.
 6. The method of claim 1 wherein in step a), said (1-84) PTHis from a species selected from the group consisting of a human, a rat,a mouse, a bovine, a dog, a pig, a cat, and a monkey.
 7. The antibodyproduced by the method of claim
 1. 8. A test kit for detecting bioactive(1-84) PTH utilizing the antibody of claim
 7. 9. The antibody of claim 7wherein said antibody is further provided with a detectable moiety.